Genomic Complexity Predicts Resistance to Endocrine Therapy and CDK4/6 Inhibition in Hormone Receptor-Positive (HR+)/HER2-Negative Metastatic Breast Cancer

Abstract

Purpose: Clinical biomarkers to identify patients unlikely to benefit from CDK4/6 inhibition (CDK4/6i) in combination with endocrine therapy (ET) are lacking. We implemented a comprehensive circulating tumor DNA (ctDNA) analysis to identify genomic features for predicting and monitoring treatment resistance.

Experimental design: ctDNA was isolated from 216 plasma samples collected from 51 patients with hormone receptor-positive (HR+)/HER2-negative (HER2-) metastatic breast cancer (MBC) on a phase II trial of palbociclib combined with letrozole or fulvestrant (NCT03007979). Boosted whole-exome sequencing (WES) was performed at baseline and clinical progression to evaluate genomic alterations, mutational signatures, and blood tumor mutational burden (bTMB). Low-pass whole-genome sequencing was performed at baseline and serial timepoints to assess blood copy-number burden (bCNB).

Results: High bTMB and bCNB were associated with lack of clinical benefit and significantly shorter progression-free survival (PFS) compared with patients with low bTMB or low bCNB (all P < 0.05). Dominant APOBEC signatures were detected at baseline exclusively in cases with high bTMB (5/13, 38.5%) versus low bTMB (0/37, 0%; P = 0.0006). Alterations in ESR1 were enriched in samples with high bTMB (P = 0.0005). There was a high correlation between bTMB determined by WES and bTMB determined using a 600-gene panel (R = 0.98). During serial monitoring, an increase in bCNB score preceded radiographic progression in 12 of 18 (66.7%) patients.

Conclusions: Genomic complexity detected by noninvasive profiling of bTMB and bCNB predicted poor outcomes in patients treated with ET and CDK4/6i and identified early disease progression before imaging. Novel treatment strategies including immunotherapy-based combinations should be investigated in this population.

Figure 1.

Overview of study design. A, Study schema. B, Sample collection timepoints for PredicineCNB and PredicineWES+ profiling. C, Next-generation sequencing process. BL, baseline; C1D15, cycle 1 day 15; C2D1, cycle 2 day 1; cfDNA, cell-free DNA; gDNA, genomic DNA; PD, progressive disease; QC, quality control.

Figure 2.

High bTMB at baseline is associated with poor patient outcomes. A, Distribution of bTMB scores across 50 baseline patient samples sequenced by PredicineWES+. High bTMB scores were significantly associated with (B) lack of clinical benefit (CB) defined as progressive disease (PD) within 6 months and (C) the presence of ESR1 mutations at baseline (Wilcoxon test; FDR P = 0.016). D, Clinical classification of endocrine resistance per ESMO 2020 guidelines did not predict bTMB, although there were a greater number of patients with high bTMB in the endocrine-resistant cohort (Kruskal–Wallis test). The association of high bTMB with significantly shorter PFS was observed using multiple cutoffs for bTMB including the median (E), third quartile (F), or bTMB scores of 10 mutations/megabase (G; log rank test). H, Within the endocrine-resistant cohort, high bTMB scores were significantly associated with shorter PFS (log-rank test).

Figure 3.

High correlation between bTMB determined from WES and a 600-gene targeted sequencing panel. bTMB levels generated from 50 baseline samples using the PredicineWES+ and PredicineATLAS sequencing assays were highly correlated (Spearman rank test).

Figure 4.

High bTMB at baseline is associated with dominant APOBEC mutational signatures and upregulation of specific oncogenic signaling pathways. A, Dominant APOBEC mutational signatures (SBS2 and SBS13) were enriched in patients with high (N = 13) versus low (N = 37) bTMB scores (defined by upper quartile cutoff). Dominant SBS5 (unknown) and SBS6 (defective DNA mismatch repair signatures) were observed across patients with high and low bTMB scores. B, The proportion of dominant APOBEC signatures was significantly greater in patients with high bTMB scores versus low bTMB scores (Fisher exact test). C, The median bTMB score was significantly higher in patients with dominant APOBEC signatures versus other signatures (Wilcoxon rank sum test).

Figure 5.

Specific genomic alterations are enriched at disease progression. A, Heatmaps comparing the most frequently altered genes detected at progression versus baseline by PredicineWES+ (N = 28). Color coded bars below each heatmap indicate the level of bTMB, and the bar graph above the progression heatmap indicates PFS in months for each patient. Blue arrows denote patients with dominant APOBEC signatures. B, Enrichment at progression was observed for previously reported gene alterations implicated in resistance to endocrine and/or CDK4/6i therapy including a significantly higher frequency of RB1 alterations (P = 0.04; McNemar test), and nonsignificant enrichment in AR, AURKA, CCND1, CDKNKA, ESR1, FGFR, MYC, PTEN, and TP53 alterations. In addition, nonsignificant enrichment of gene alterations not commonly linked to ET + CDK4/6i resistance such as BRCA2, CBL, CDH, KMT2D, MUC12, and PREX2 was observed at progression. Two of the enriched gene alterations (MUC12 and PREX2) were not covered by the targeted PredicineATLAS panel and were detected only through comprehensive WES.

Figure 6.

bCNB scores predict patient outcomes and increase before radiographic detection of clinical progression. bCNB scores were generated for all 51 patients at baseline and during treatment via sequencing with PredicineCNB. A, Baseline bCNB scores were significantly higher in patients who experienced progression within 6 months (without clinical benefit; Wilcoxon test). B, High baseline bCNB scores were significantly associated with shorter PFS (log rank test). C, Serial analysis of bCNB during treatment revealed decreases at C1D15 and/or C2D1, followed by increases that preceded imaging detection of progressive disease in 12 of 18 (66.7%) patients for whom staging blood samples were analyzed.